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H Chong

Publications and source records attributed to H Chong.

At least 19 recordsLinked to original sources
PubMed2001

Function of the Rho family GTPases in Ras-stimulated Raf activation.

Ras plays an essential role in activation of Raf kinase which is directly responsible for activation of the MEK-ERK kinase pathway. A direct protein-protein interaction between Ras and the N-terminal regulatory domain of Raf is critical for Raf activation. However, association with Ras is not sufficient to activate Raf in vitro, indicating that Ras must activate some other biochemical events leading to activation of Raf. We have observed that RasV12Y32F and RasV12T35S mutants fail to activate Raf, yet retain the ability to interact with Raf. In this report, we showed that RasV12Y32F and RasV12T35S can cooperate with members of the Rho family GTPases to activate Raf while alone the Rho family GTPase is not effective in Raf activation. A dominant negative mutant of Rac or RhoA can block Raf activation by Ras. The effect of Rac or Cdc42 can be substituted by the Pak kinase, which is a direct downstream target of Rac/Cdc42. Furthermore, expression of a kinase inactive mutant of Pak or the N-terminal inhibitory domain of Pak1 can block the effect of Rac or Cdc42. In contrast, Pak appears to play no direct role in relaying the signal from RhoA to Raf, indicating that RhoA utilizes a different mechanism than Rac/Cdc42. Membrane-associated but not cytoplasmic Raf can be activated by Rac or RhoA. Our data support a model by which the Rho family small GTPases play an important role to mediate the activation of Raf by Ras. Ras, at least, has two distinct functions in Raf activation, recruitment of Raf to the plasma membrane by direct binding and stimulation of Raf activating kinases via the Rho family GTPases.

Animals↗
PubMed2001

Positive and negative regulation of Raf kinase activity and function by phosphorylation.

Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function.

Amino Acid Sequence↗
PubMed2000

Structure and expression of a membrane component of the inhibin receptor system.

The purification and cloning of a membrane-anchored proteoglycan with affinity for inhibin A are described. Bovine pituitary membranes were isolated, and membrane-anchored proteins were solubilized and used as an enriched source of inhibin binding protein. The extract was passed over an inhibin A affinity column, and a protein, designated p120, was identified as an inhibin-binding moiety. A partial amino acid sequence was determined for the protein, which matched two human complementary DNAs (cDNAs) in the database. The full-length cDNA predicts a 1336-amino acid glycoprotein. Full-length p120-encoding cDNAs were isolated from human testis RNA and cloned into expression vectors. Two p120 messenger RNA transcripts of 4.6 kb and 2 kb are detected in rat pituitary by RNA blot analysis. Similar analysis of rat testis RNA revealed transcripts of identical molecular mass, albeit at lower abundance. To determine the cellular localization of p120 in pituitary and testis, an antibody directed against the predicted extracellular domain of the protein was generated and used in an immunohistochemical analysis of thin tissue sections. p120 immunostaining is coincident with FSHbeta immunopositive gonadotrope cells in rat pituitary. p120 staining is intense in the testicular Leydig cells, which bind iodinated inhibin but not iodinated activin. In summary, an inhibin-binding protein has been isolated that is produced in tissues that are targets of inhibin action.

Activin Receptors↗
PubMed1999

Adoptive transfer of immature dendritic cells with autologous or allogeneic tumor cells generates systemic antitumor immunity.

Dendritic cells (DCs) are potent antigen-presenting cells that are capable of priming systemic antitumor immune responses in animal tumor models. However, many of the model tumor systems tested need definition of the specific tumor antigens involved. To use DCs in situations that are more relevant to the majority of human cancers, where the antigens are unknown, we have tested the adoptive transfer of immature DCs in mouse colorectal and melanoma models of varying immunogenicity but with undefined antigens. When DCs admixed with a syngeneic primary tumor inoculum were seeded s.c., the growth of the primary tumor was unchanged; however, if the primary tumor was then surgically excised and the animal was rechallenged with the same tumor, significant protection (75%) was generated when DCs were present in the original primary inoculum of a moderately immunogenic colorectal model (CMT93tk). This effect was not observed when a nonimmunogenic melanoma (B16) was tested in an identical protocol. Next, DCs were injected directly into 6-9-mm established tumors; again, protection (55%) was achieved against a secondary tumor challenge following excision of the primary, but only in the CMT93tk model of moderate immunogenicity. To increase the clinical relevance of this approach still further, we tested irradiated allogeneic K1735 melanoma cells mixed with syngeneic DCs as a vaccine against subsequent challenge with the poorly immunogenic syngeneic melanoma B16. The allogeneic vaccine alone was ineffective, but when admixed with DCs, a significant number of animals rejected a subsequent B16 challenge, suggesting that DCs are able to prime an immune response against melanoma antigens shared between K1735 and B16. The generation of systemic antitumor immunity by adoptive transfer of DCs has significant clinical potential because it is technically straightforward and does not require the definition of specific tumor antigens.

Animals↗
PubMed1999

Regulation of transforming growth factor beta1 by nitric oxide.

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.

Adenocarcinoma↗
PubMed1999

Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages.

Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

Animals↗
PubMed1999

Anti-tumour activity against B16-F10 melanoma with a GM-CSF secreting allogeneic tumour cell vaccine.

Genetic modification of tumour cells with the GM-CSF encoding gene renders these cells more potent, as autologous tumour cell vaccine, than their wild-type counterparts. However, autologous vaccines are impractical for wide-scale clinical use and we have therefore investigated the efficacy of the GM-CSF genetic modification approach with an allogeneic whole cell tumour vaccine. In this report, we show that the allogeneic K1735-M2 (H-2k) melanoma cell vaccine induces a specific protective anti-tumour response against the syngeneic B16-F10 (H-2b) melanoma tumour in C57BL/6J mice. In vitro T cell work demonstrated that vaccination of animals with the allogeneic cell vaccine generated cytotoxic T cells specific for the autologous tumour. In vivo T cell subset depletion experiments also illustrated that this anti-tumour effect was mediated by both CD4+ve and CD8+ve T cells, suggesting that the allogeneic vaccine may operate through the 'cross-priming' phenomenon whereby tumour antigens are processed and presented to T cells by the host's own antigen presenting cells (APC). Thus, we transduced K1735-M2 cells with a GM-CSF expressing retroviral vector and showed anti-tumour activity of the GM-CSF secreting K1735-M2 cells as a therapeutic vaccine against the syngeneic B16-F10 tumour. Our data imply that GM-CSF genetically modified allogeneic whole cell tumour vaccines could be successful in the clinic. In addition, more potent combination gene therapy strategies could be tested using this therapeutic allogeneic vaccine model.

Animals↗
PubMed1998

Expression of B7 co-stimulatory molecules by B16 melanoma results in a natural killer cell-dependent local anti-tumour response, but induces T-cell-dependent systemic immunity only against B7-expressing tumours.

In an attempt to enhance the anti-tumour immune response, the co-stimulatory molecules B7-1 or B7-2 were expressed on the surface of B16 melanoma cells. B7-expressing tumours grew more slowly in both syngeneic immunocompetent mice and athymic T cell-immunodeficient nude mice. The delay in growth of B7-expressing tumours was dependent on natural killer (NK) cells, as reductions in tumour growth rates were minimized in mice depleted of NK cells. Systemic immunity to B16 melanoma was examined by vaccination with irradiated tumour cells. Inoculation with irradiated B16 B7-1 cells failed to protect against a subsequent challenge with live parental B16 cells, but conferred partial protection against challenge with live B16 B7-1 cells. In contrast to the local anti-tumour reaction, this protective response was dependent on T cells. The results presented here reveal some of the mechanisms involved in the in vivo response to a poorly immunogenic tumour modified to express co-stimulatory molecules.

Animals↗
PubMed1998

Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte-macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity.

Previously, we showed that expression of B7-1 in CMT93 murine colorectal tumour cells inhibited their growth in immunocompetent animals. However, this did not result in any significant increase in systemic protective immunity, relative to that elicited by the parental tumour. To potentiate the effects of B7-1 on systemic immunity. Interleukin-12 (IL-12) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was co-expressed with this molecule. These combinations of immunostimulatory molecules were effective in eliciting systemic immunity. We also show that expression of B7-2 led to a local antitumour response as well as significantly raised systemic immunity. In another tumour model. K1735 minutes melanoma, which is moderately immunogenic, tumours secreting GM-CSF alone were as effective as the parental tumours in generating protective immunity. Previously, we described the deleterious effect of B7-1 expression on protective immunity. Co-expression of GM-CSF did not counteract this consequence of B7-1 expression. Expression of IL-12 was extremely effective in causing rejection of inoculated tumour cells, but evoked only minimal protective systemic immunity. These results suggest that combing costimulatory molecules and cytokines may be a useful therapeutic approach in some, but not all, tumours.

Animals↗
PubMed1998

Rapid adenoviral transduction of freshly resected tumour explants with therapeutically useful genes provides a rationale for genetic immunotherapy for colorectal cancer.

To develop protocols for the molecular immunotherapy of colorectal cancer, we compared the efficacy of three separate classes of therapeutic genes to induce antitumour responses in a murine colorectal cell model. Thus, the effects of two cytokines (IL-2 and GM-CSF) were compared with those of a costimulatory gene (B7.1) and a suicide gene (HSVtk). The rank order of efficacy against primary tumour growth was HSVtk[GCV], B7.1 > puro, IL-2 > GM-CSF, neo whereas the order of efficacy in inducing antitumour immunity was GM-CSF, IL-2, > B7.1, HSVtk[GCV] > puro, neo in a prophylactic vaccination model. To exploit these data in a clinically relevant and realistic way, we also demonstrated that colorectal tumours can reproducibly be explanted and established in short-term culture. Finally, a rapid transduction protocol has been developed by which, using adenoviral vectors, as many as 90% of the cells in these fresh tumour explants can be engineered to express high levels of the clinically relevant genes (GM-CSF or IL-2) within 1-2 weeks of surgery. Adenovirus-mediated gene delivery was reproducibly and significantly more efficient than retroviral transduction using the MFG-beta-Gal retroviral vector over the time-frame of importance for vaccination. Hence, combination of the animal model data with the ex vivo modification protocol suggests that vaccination of colorectal patients of the appropriate stage will be possible and effective.

Adenoviridae↗
PubMed1998

A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences.

Previously we reported the presence of a replication-competent retrovirus in supernatant from a vector-producing line derived from a widely used split-function amphotropic packaging cell line. Rigorous routine screening of all retroviral stocks produced in our laboratory has not, previously or since, indicated the presence of such a virus. Replication-competent retroviruses have never previously been used in our laboratory, and stringent screening of all routinely used cell lines has not revealed the presence of any helper viruses. Therefore, it is highly unlikely that this virus represents an adventitious cross-contaminant or had been imported unknowingly with our cell line stocks. PCR studies with DNA from infected cell lines and Northern blot analysis and reverse transcriptase PCR with RNA from infected cells suggest that the helper virus arose by recombination events, at sites of partial homology, between sequences in the vector, one of the packaging constructs, and endogenous retroviral elements. These recombinations were not present in stocks of the packaging cell line or in an initial stock of the vector-producing line, indicating that these events occurred while the vector-producing line was being passaged for harvest of supernatant stocks.

Animals↗
PubMed1998

Can immunotherapy by gene transfer tip the balance against colorectal cancer?

Gene therapy, in particular the transfer of genes encoding immunostimulatory molecules (cytokines and costimulatory molecules) as well as selectively cytotoxic enzymes and DNA vaccination, has the potential of enhancing cell mediated immune responses against tumours including those of colorectal origin. Genes can be transferred using viral vectors either to cultured tumour cells in vitro that can be returned to the patient as a "cancer vaccine", or directly to tumour cells in vivo. Vaccination with DNA constructs expressing specific tumour antigens characteristic of colorectal neoplasia can trigger immune recognition and destruction of tumour cells. The aim is to tip the balance from protumour to antitumour mechanisms by generating a local immune response and systemic antitumour immune memory to destroy metastases. Studies in murine models, combined with human studies, show that such approaches could become an adjunct to current treatments for human colorectal cancer in the near future.

Colorectal Neoplasms↗
PubMed1998

[Measurement of contraction force of pilot's neck muscles].

In order to discuss the function of contraction force of pilot's neck region. Contraction force of pilot's neck region was measured in 22 volunteers aged 18-35 years. The mean contraction force is 102-396 N, the mean contraction time is 28 s. Authors think that training for pilot's neck muscular power is essential.

Adolescent↗
PubMed1997

A family of bicistronic vectors to enhance both local and systemic antitumor effects of HSVtk or cytokine expression in a murine melanoma model.

The herpes simplex virus-thymidine kinase/ganciclovir (HSVtk/GCV) system produces both direct and immune-mediated tumor cell killing. Here, we compare the efficacy of HSVtk/GCV with cytokines, alone and in combination, on the tumorigenicity and immunogenicity of B16 cells. With respect to single gene modifications, only HSVtk/GCV, or high-level interleukin-2 (IL-2) secretion, completely prevented tumor growth, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) generated the best levels of long-term systemic protection. To augment both local killing and immune activation, we constructed bicistronic constructs that express HSVtk and a cytokine within the same cell. Co-expression of HSVtk with IL-2 or GM-CSF enhanced the local antitumor activity of any gene alone. In a tumor-prevention model, HSVtk killing, in an environment preprimed with GM-CSF, generated the best long-term immune protection. However, in a short-term therapy model, continued IL-2 expression was most effective against 3-day established tumors. This probably reflects differences in the activities of IL-2 and GM-CSF in generating short-term, nonspecific immune stimulation compared to long-term immunological memory, respectively. As a prelude to in vivo delivery experiments, we also demonstrated that these bicistronic cassettes can be packaged normally into retroviral (5 x 10(5) virus/ml from pooled populations) and adenoviral vectors (5 x 10(9) virus/ml) and function as predicted within virally infected cells. This family of bicistronic vectors can be used to stimulate synergy between suicide and cytokine genes, overcomes the problems of delivering two genes on separate vectors, and should allow easier preparation of vectors for the delivery of multiple genes to patients' tumor cells.

Adenoviridae↗
PubMed1997

Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression.

Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, anti-tumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk+ tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4+ and CD8+ T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk+ cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo.

Animals↗
PubMed1997

The uterine myometrium is a target for increased levels of activin A during pregnancy.

Activin A is a dimeric protein hormone that regulates numerous cellular functions. A clear physiological role for this molecule in pregnancy is suggested by previous studies in the human, wherein activin A rises dramatically as women approach parturition. To determine whether the rodent is a suitable animal model for further studies of activin action during pregnancy, the serum concentration of activin A was measured in pregnant rats. Activin A was detected in the serum of pregnant rats, beginning on day 12, and the serum concentration rose progressively through gestation (22-fold) and dramatically (140-fold) in labor. The potential target tissues for circulating activin were then identified in two ways. First, iodinated activin was injected into pregnant rats, and the tissues targeted by labeled ligand were identified in vivo. A tissue targeted by activin A in the pregnant rat was the uterine myometrium. To determine the ligand specificity of the uterine myometrial cells, the uteri of pregnant rats were collected and analyzed by in situ ligand binding. 125I-activin A binding was specific for the uterus myometrium, and the ligand binding was competed by unlabeled activin A but not by inhibin A. This result suggests that the receptor in this tissue compartment is an activin-specific receptor. The production of abundant activin A and the ability of exogenous ligand to target the myometrium of the uterus provides a pathway by which activin could regulate uterine function during pregnancy.

Activins↗
PubMed1996

Expression of co-stimulatory molecules by tumor cells decreases tumorigenicity but may also reduce systemic antitumor immunity.

Many tumor cells do not express co-stimulatory molecules, and this may account, in part, for their poor ability to stimulate T cells directly. One strategy to enhance immune recognition would be to express such molecules on the tumor cell. Here, we show that expression of a member of the B7 family of co-stimulatory molecules by CMT93 murine colorectal tumor or 1735 murine melanoma cells resulted in a local antitumor response in immunocompetent mice. The antitumor effect was diminished in athymic nude mice, indicating that T cells played an important part in this response. The ability of the B7-expressing tumor cells to generate systemic protective immunity was investigated by excision of tumors that developed from the initial inoculation followed by rechallenge with parental tumor cells. CMT93 is a poorly immunogenic tumor and no significant systemic immunity was elicited by the expression of B7-1 in these cells. 1735 melanoma is a mildly immunogenic tumor. Unexpectedly, the systemic immunity obtained with 1735 tumors expressing B7-1 or B7-2 was weaker than that generated by parental 1735 cells (p < 0.001, stratified logrank test), even when coexpression of interferon-gamma in the B7-1 cells produced high levels of surface MHC class I expression. These results suggest that some caution is appropriate when considering the use of these molecules in the gene therapy of cancer.

Animals↗